Circadian rhythms are fundamental features of many if not all organisms on Earth. Among animals, the molecular mechanisms underlying these rhythms are remarkably conserved. Yet, outside animals, known mechanisms among the few model systems are disparate, which is not surprising due to the billions of years of separation among these taxa. Among prokaryotes, only one species has been shown to exhibit circadian rhythms, the cyanobacterium Synechococcus elongatus, whose clock derives from cycles of autophosphorylation and phosphatase activity in key clock proteins Kai A,B and C. We have discovered a new, biomedically important model bacterium, Enterobacter aerogenes, which expresses a temperature-compensated circadian clock, which is synchronized by the gut hormone melatonin and is entrained by cycles of temperature, using swarming behavior and motA-driven luciferase bioluminescence. We propose to fully characterize this exciting new clock system. We will determine whether the clock system regulates multiple physiological processes in parallel by asking whether gene expression with different promoter elements and luciferase are expressed rhythmically and/or respond to melatonin similar to motA-driven rhythms. We will determine whether melatonin is a true Zeitgeber for this clock, and we will characterize the effects of temperature cycles, which entrain this clock, testing the hypotheses that host melatonin secretion and/or TB synchronize this enteric bacterium. Secondly, we will knock out candidate genes that bear molecular similarity to Kai proteins, characterize these knockouts, and complement those that exhibit clock phenotypes. In addition, we will conduct a broad mutant screen, searching for clock mutants. Thirdly, we will characterize the biological activity, clock function and melatonin sensitivity of all knockout and mutant strains discovered here. If this clock does indeed share a common ancestor with the cyanobacterial clock, the data have broad and important evolutionary significance, placing the evolution of the circadian clock before the emergence of O2 generating photosynthesis some 3.5 billion years ago. In addition, these observations point to an integrated circadian organization in which master pacemakers in the brain synchronize peripheral oscillators, which in turn may regulate aspects of the microbiota through entrainment of the bacterial clock. This has important implications for biomedical science and therapeutics.